Extracellular vesicles comprising recombinant lysosomal transmembrane protein

ABSTRACT

The present invention provides compositions and methods for providing factor replacement therapy. In particular, the present invention provides replacement therapy for subjects suffering from cystinosis.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. patent applicationSer. No. 12/842,853, filed Jul. 23, 2010, which claims priority to U.S.Provisional Application 61/228,327, filed Jul. 24, 2009, each of whichis herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides compositions and methods for providingfactor replacement therapy. In particular, the present inventionprovides replacement therapy for subjects suffering from cystinosis.

BACKGROUND OF THE INVENTION

Cystinosis has been known since the first decade of the last century.Small, pale children who died of wasting and whose organs were shown tobe riddled with microscopic crystals were described by Abderhalden in1903. He termed the condition “Familiare Cystindiathese” (Thoene. MolGenet Metab. 2007, herein incorporated by reference in its entirety).The disease has been conflated with the unrelated condition cystinuria,which is an extracellular disease of renal transport which results inexcess cystine urinary excretion, leading to cystine stones in theurinary tract (Palacin et al., eds. The Metabolic and Molecular Bases ofInherited Disease, 8th edition, McGraw Hill, 4909-4932, 2001, hereinincorporated by reference in its entirety), while cystinosis is anintracellular condition that results from defective lysosomal cystinetransport, leading to lysosomal cystine storage. The lysosomal cystinetransporter, cystinosin, is encoded by CTNS, located at 17p13.3, andfunctions to move cystine from the lysosomal interior to the cytosol,where it can be reused for GSH and protein synthesis. Many mutationshave been described at this locus, however a large 57 kb deletionaccounts for about half the cases descended from west European parents.The clinical phenotype in nephropathic cystinosis is relatively unique:renal Fanconi syndrome with salt, water, glucose, amino acid and othersmall molecule losses; crystalline keratopathy and salt and pepperretinopathy; short stature and failure to thrive; photophobia; andultimately renal death by age 10 years. Other elements includehypothyroidism, and later in life, muscle weakness, esophagealdysmotility, and diabetes. It produces the renal Fanconi syndrome withsalt, water and other small molecule wasting, deficient bonemineralization, short stature, failure to thrive, and later in life,hypothyroidism, muscle wasting and weakness, esophageal dysmotility,pancreatic deficiency, and pulmonary involvement. It is treatedsymptomatically with salt and water replacement, and specifically, withcysteamine, which causes depletion of the stored lysosomal cystine whichis the biochemical hallmark of the condition and which results frommutations in the lysosomal cystine transporter, cystinosin. Therapy ofcystinosis has taken several forms, although none is currentlysatisfactory. Initially recognition of the peril these children are infrom the risk of dehydration and electrolyte imbalance led tosymptomatic water and electrolyte replacement therapy. Subsequently,chronic dialysis and then renal transplantation were used to compensatefor the renal failure. Specific therapy for cystinosis was achieved in1994 when the FDA approved cysteamine bitartrate as Cystagon for thetreatment of cystinosis. This drug causes depletion of cystine fromcystinotic lysosomes by forming a mixed disulfide with cystine whichresembles lysine and which can thus exit lysosomes on the intact lysinetransporter. Unfortunately, the structure of cysteamine includes a freethiol group, thus the drug has the odor and taste of rotten eggs, afeature of concern both to parents administering the treatment tochildren, and to patients themselves who may find the offensive odor tobe socially debilitating. Although cysteamine has been FDA approvedsince 1994, there are problems with this therapy: 1) It does not preventrenal failure, but merely delays the onset, for most patients 2) itsrepugnant thiol odor and taste causes both gastrointestinal andcompliance problems and social concerns in children as they reachadolescence. The latter issue causes some patients to forgo ordiscontinue treatment, with the expected concomitant health consequencesresulting therefrom. For these reasons, a superior form of treatmentthat averts renal failure indefinitely, and which lacks the thiol odorand taste of cysteamine would be highly desirable.

Gene therapy has been proposed to treat a variety of conditions,including inborn errors of metabolism, however serious reactions to AVand AAV vectors has lead to questioning in the field as whether theseagents can be safely employed in patients. Gene therapy, long the hopeof patients with many diseases, both genetic and otherwise, hascontinuing issues of safety and efficacy. The safety concerns relate tointegration of the trans genes in locations that disrupt the function ofcritical tumor supressors, or other vital sequences. Other side effectsinclude serious or fatal allergic reactions to the large number ofvirions which must be administered in the quest for stable geneexpression that will have a salutary impact on phenotype. A death wasreported in July, 2007 in an arthritis patient receiving gene therapyusing an AAV vector, which was hoped to be safer than the AV vectorinvolved in the Geissinger death in 1999.

SUMMARY

In some embodiments, the present invention provides compositions andmethods for treating factor deficiency (e.g. cystinosin deficiency) in asubject. In some embodiments, the present invention providescompositions and methods for depleting cystine from lysosomes (e.g.cystinotic lysosomes). In some embodiments, the present inventionprovides compositions comprising a cystine depletion factor (CDF) andmethods of preparation (e.g. transfection, expression, purification,etc.) and use (e.g. therapeutic administration, prophylacticadministration, etc.) thereof. In some embodiments, the presentinvention provides compositions comprising a cystinosin replacementfactor and methods of preparation (e.g. transfection, expression,purification, etc.) and use (e.g. therapeutic administration,prophylactic administration, etc.) thereof. In some embodiments, thepresent invention provides compositions comprising cystinosin andmethods of preparation (e.g. transfection, expression, purification,etc.) and use (e.g. therapeutic administration) thereof. In someembodiments, the present invention provides compositions and methods fortreating cystinosis in a subject (e.g. administration of CDF). In someembodiments, the present invention provides exosomes (e.g. comprisingcystinosin) for use in treating a disease or disorder (e.g. cystinosis).

In some embodiments, the present invention provides compositionscomprising a cystine depletion factor (CDF). In some embodiments, theCDF reduces the level of cystine in lysosomes. In some embodiments, theCDF reduces the level of cystine in cystinotic lysosomes. In someembodiments, the CDF reduces the level of cystine in lysosomes to anormal, healthy, and/or non-cystinoic level. In some embodiments, theCDF is secreted from cells expressing cystinosin. In some embodiments,the CDF functions as a replacement for cystinosin in a subject sufferingfrom cystinosis. In some embodiments, administration of the CDF to asubject suffering from cystinosis provides treatment (e.g. curativeand/or palliative) for cystinosis. In some embodiments, the CDF depletescystine from lysosomes (e.g. cystinotic lysosomes) through the samemechanism as cystinosin (e.g. endogenous cystinosin in a subject notsuffering from cystinosin). In some embodiments, the CDF depletescystine from lysosomes (e.g. cystinotic lysosomes) through a differentmechanism than cystinosin. In some embodiments, the CDF comprises one ormore small molecules, proteins, peptides, macromolecules, complexes,etc. In some embodiments, CDF comprises, consists essentially of, orconsists of cystinosin.

In some embodiments, the present invention provides a cystinosinreplacement factor (CRF). In some embodiments, the CRF functions as areplacement for cystinosin in a subject suffering from cystinosis. Insome embodiments, administration of the CRF to a subject suffering fromcystinosis provides treatment (e.g. curative and/or palliative) forcystinosis. In some embodiments, the CRF depletes cystine from lysosomes(e.g. cystinotic lysosomes) through the same mechanism as cystinosin(e.g. endogenous cystinosin in a subject not suffering from cystinosin).In some embodiments, the CRF is secreted from cells expressingcystinosin. In some embodiments, the CRF reduces the level of cystine inlysosomes. In some embodiments, the CRF reduces the level of cystine incystinotic lysosomes. In some embodiments, the CRF comprises one or moresmall molecules, proteins, peptides, macromolecules, complexes, etc. Insome embodiments, the CRF comprises, consists essentially of, orconsists of cystinosin.

In some embodiments, the present invention provides a compositioncomprising cystinosin. In some embodiments, the cystinosin is purifiedor partially purified. In some embodiments, the cystinosin is isolatedor partially isolated. In some embodiments, the cystinosin isrecombinantly expressed. In some embodiments, the cystinosin is producedin cells transfected with the CTNS gene or variants thereof. In someembodiments, the present invention provides a composition comprisingpurified and soluble recombinant cystinosin. In some embodiments, therecombinant cystinosin functions as a replacement for endogenouscystinosin in a subject suffering from cystinosis. In some embodiments,cystinosin is complexed with one or more additional, agents, compounds,peptides, macromolecules, complexes, etc.

In some embodiments, the present invention providescystinosin-expressing cells. In some embodiments, cells expressrecombinant cystinosin. In some embodiments, cells are transfected withthe gene for cystinosin (CTNS) or variants thereof. In some embodiments,cells secrete cystinosin, CRF, and/or CDF. In some embodiments, cellssecrete or otherwise generate exosomes comprising cystinosin, CRF,and/or CDF.

In some embodiments, the present invention provides compositions andmethods for treating cystinosis. In some embodiments, the presentinvention provides a method for treating cystinosis comprisingadministering an isolated portion of liquid media, within which cellsexpressing cystinosin were grown, to subjects suffering from cystinosis.In some embodiments, the isolated portion of the liquid media provides areplacement for cystinosin. In some embodiments, the isolated portion ofthe liquid media undergoes a purification step prior to administering tothe subject.

In some embodiments, the present invention provides a method fortreating cystinosis comprising administering recombinant cystinosin to asubject suffering from cystinosis. In some embodiments, the recombinantcystinosin is expressed in cells. In some embodiments, the cellscomprise Sf9 insect cells. In some embodiments, the cells are grown inliquid media. In some embodiments, the recombinant cystinosin isobtained from the liquid media. In some embodiments, the recombinantcystinosin is purified from the liquid media. In some embodiments, therecombinant cystinosin functions as a replacement for endogenouscystinosin in a subject suffering from cystinosis.

In some embodiments, the present invention provides a factor replacementtherapy comprising: a) expressing the factor in cells, wherein the cellsare grown in liquid media, and b) administering an isolated portion ofthe liquid media to a subject lacking the factor. In some embodiments,the isolated portion of said liquid media comprises elements purifiedfrom the liquid media. In some embodiments, the factor provides asupplement to the subject lacking the factor. In some embodiments, theisolated portion of said liquid media comprises compositions whichcompensate for the factor in the subject lacking the factor. In someembodiments, the factor comprises cystinosin. In some embodiments, thesubject suffers from cystinosis. In some embodiments, administering ofthe isolated portion of the liquid media provides a replacement forcystinosin in the subject. In some embodiments, the isolated portion ofthe liquid media comprises recombinant cystinosin.

In some embodiments, the present invention provides a compositioncomprising cystinosin replacement factor, wherein the cystinosinreplacement factor functions as a replacement for endogenous cystinosinin a subject suffering from cystinosis. In some embodiments, thecystinosin replacement factor is configured to deplete cystine fromcystinotic lysosomes. In some embodiments, the cystinosin replacementfactor is obtained from a liquid media in contact with cells expressingrecombinant cystinosin. In some embodiments, the cystinosin replacementfactor comprises recombinant cystinosin. In some embodiments, all or aportion of the recombinant cystinosin is encapsulated within one or moreexosomes. In some embodiments, the composition further comprises apharmaceutically acceptable buffer.

In some embodiments, the present invention provides a method fortreating cystinosis, comprising: administering an isolated portion ofliquid media, within which cells expressing cystinosin were grown, tosubjects suffering from cystinosis. In some embodiments, the isolatedportion of liquid media comprises a cystine depletion factor. In someembodiments, the isolated portion of liquid media comprises asedimentable fraction of the liquid media. In some embodiments, thecystine depletion factor comprises exosomes. In some embodiments, theexosomes comprise cystinosin. In some embodiments, the isolated portionof the liquid media undergoes one or more purification steps prior toadministering to said subject. In some embodiments, the isolated portionof the liquid media is combined with a pharmaceutically acceptablebuffer. In some embodiments, the isolated portion of the liquid mediaprovides a replacement for endogenous cystinosin.

In some embodiments, the present invention provides a method fortreating cystinosis comprising administering recombinant cystinosin to asubject suffering from cystinosis. In some embodiments, the recombinantcystinosin is expressed in cells. In some embodiments, the cellscomprise Sf9 cells. In some embodiments, the cells are grown in liquidmedia. In some embodiments, the recombinant cystinosin is purified fromsaid liquid media. In some embodiments, the recombinant cystinosinsedimented by centrifugation. In some embodiments, the recombinantcystinosin is encapsulated in exosomes. In some embodiments, therecombinant cystinosin functions as a replacement for endogenouscystinosin in a subject suffering from cystinosis.

In some embodiments, the present invention provides compositions andmethods for factor replacement therapy. In some embodiments, a factor isproduced and administered to a subject deficient in the factor as atreatment for the deficiency. In some embodiments, the factor isexpressed in cells. In some embodiments, the factor is a protein orpeptide. In some embodiments, the factor is secreted from cells inexosomes. In some embodiments, the factor is encapsulated in exosomes.In some embodiments, the present invention provides exosomes comprisinga replacement factor. In some embodiments, the present inventionprovides administering exosomes comprising a replacement factor to asubject deficient in that subject as a treatment for factor deficiencyand/or related conditions and/or symptoms.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary and detailed description is better understood whenread in conjunction with the accompanying drawings which are included byway of example and not by way of limitation.

FIG. 1 shows an agarose gel of PCR identification of the Bacmidcontaining the cloned human cystinosin gene; Lane 1: negative control(Bacmid alone), Lane 2: unsuccessful cloning, Lane 3: human cystinosingene in pFastBacHTA, and Lane 4. positive control (TGF-beta inpFastBac-GST).

FIG. 2 shows Western blot analysis of P0 Cell pellet; Lane 1: whole celllysate of P0 cell pellet, Lane 2: conditioned medium of P0, and Lane 3:whole cell lysate of Bacmid.

FIGS. 3A-D show MOI optimization: 10 ml 2*10⁶/ml sf9 cells were infectedat the MOI of 1, 2, 5, 10 and 20 separately, and each 1 ml cell pelletwas collected 1 dpi, 2 dpi, 3 dpi and 4 dpi. The cells were analyzed bySDS-PAGE and western blot.

FIGS. 4A-B show an (A) SDS-PAGE and (B) Western blot analysis of aprotein solubility test: 200 ml infected sf9 cells were collected andwashed by PBS once; the cells were suspended with 20 mM Tris.HCl, 1 mMDTT, 1% NP-40, pH8.0 on ice for 45 min and sonicated. The supernatantand the pellet were separated at the speed of 12,000 rpm for 10 min at4° C. SDS-PAGE (A) and western blot analyzed the solubility of theprotein. Lane 1: Sf9 bacmid, Lane 2: whole cell lysate, Lane 3: cytosoltreated with 20 mM Tris-HCl 1 mM DTT 1% NP-40 pH8, and Lane 4: pellet.

FIGS. 5A-B show a (A) SDS-PAGE and (B) Western blot analysis of anextraction test in which cell pellet was treated with Buffer A (10 mMHEPES-KOH, pH7.9 at 4° C., 1.5 mM MgCl2, 10 mM KCl, 1 mM PMSF) on icefor 45 min, the supernatant and the pellet was separated, the buffer C(20 mM HEPES-KOH, pH7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 1.5 mMMgCl2) then was followed. The obtained pellet from Buffer C was aliquotinto 6 parts, and treated with the buffers (20 mM Tris.HCl, 2%TritonX-100 1% deoxycholate, 1 mM beta-mercaptoethanol, 5% Glycerol) ofdifferent pH and different concentration of NaCl. (A) Lane 1: whole celllysate of Bacmid, Lane 2: pellet treated with 20 mM Tris-HCl 1 mM DTT 1%NP-40 pH 8, Lane 3: cytosol treated with Buffer A, Lane 4: cytosoltreated with Buffer C, Lane 5: cytosol treated with 150 mM NaCl pH 7.5,Lane 6: cytosol treated with 150 M NaCl pH 10, Lane 7: cytosol treatedwith 500 mM NaCl pH 7.5, Lane 8: cytosol treated with 500 mM NaCl pH 10,Lane 9: cytosol treated with 1M NaCl pH 7.5, Lane 10: cytosol treatedwith 1M NaCl pH 10. (B) Lane 1: whole cell lysate of Bacmid, Lane 2:whole cell lysate of 44783, Lane 3: cytosol treated with 20 mM Tris-HCl1 mM DTT 1% NP-40 pH 8, Lane 4: pellet treated with 20 mM Tris-HCl 1 mMDTT 1% NP-40 pH 8, Lane 5: cytosol treated with Buffer A, Lane 6:cytosol treated with Buffer C, Lane 7: cytosol treated with 150 mM NaCl,pH 7.5, Lane 8: cytosol treated with 150 mM NaCl, pH 10, Lane 9: cytosoltreated with 500 mM NaCl, pH 7.5, Lane 10: cytosol treated with 500 mMNaCl, pH 10, Lane 11: cytosol treated with 1M NaCl, pH 7.5, Lane 12:cytosol treated with 1M NaCl pH 10. The SDS-PAGE (A) and Western blot(B) result shows the target protein was not dissolved in any buffer.

FIG. 6 shows Western blot of extraction test with urea: infected cellswere suspended with 50 mM Tris.HCl, 300 mM NaCl, 2% Triton X-100, and pH8.0 for 45 min on ice, and then treated with 8M urea, 50 mM Tris-HCl,500 mM NaCl, pH 8.0 for about half an hour. Lane 1: whole cell lysate ofBacmid, Lane 2: whole cell lysate of 44783, Lane 3: supernatant treatedwith 8M urea, Lane 4: pellet treated with 8M urea. The Western blotshows the protein still exists in the pellet.

FIGS. 7A-B show (A) SDS-PAGE and (B) Western blot analysis of proteinextraction with SKL and thiourea: infected cells were treated with 20 mMTris-HCl 0.03% SKL pH8.0; the infected cell was then treated with 20 mMTris-HCl 0.5% CHAPS 20 mM DTT, 0.4% ABS14, 2M thiourea, 6M urea pH8.0,the result shows that the protein in insoluble: Lane 1: whole celllysate, Lane 2: pellet treated with 20 mM Tris-HCl 0.03% SKL pH 8, Lane3: cytosol treated with 20 mM Tris-HCl 0.03% SKL pH 8, Lane 4: pellettreated with 20 mM Tris-HCl 0.5% CHAPS 20 mM DTT 0.4% ABS14 2M thiourea6M urea pH 8, Lane 5: cytosol treated with 20 mM Tris-HCl 0.5% CHAPS 20mM DTT 0.4% ABS142M thiourea 6M urea pH 8, and Lane 6 (B only): Sf9bacmid.

FIGS. 8A-B show (A) SDS-PAGE and (B) Western blot analysis of Gu.HCltreatment and purification. Infected cells were extracted with 20 mMTris-HCl 500 mM NaCl 2% TritonX-100 1% deoxycholate 1 mM BME 5% GlycerolpH 7.5 once, and then treated with 6M Gu.HCl. The supernatant was loadedon the Nickel column, eluted with 20 mM imidazole and 250 mM imidazole.The solutions obtained were diazlyzed against 20 mM Tris-HCl, 150 mMNaCl, 10% glycerol, but the precipitate produced. Protein was elutedwith 20 mM imidazole, but was precipitated when renaturing. (A) Lane 1:whole cell lysate of Bacmid, Lane 2: whole cell lysate of 44783, Lane 3:cytosol treated with 20 mM Tris.HCl 500 mM NaCl 2% TritonX-100 1%deoxycholate, 1 mM BME 5% Glycerol pH 7.5, Lane 4: cytosol treated withPBS, Lane 5: pellet treated with 6M Gu.HCl, Lane 6. Eluted with 20 mMTris.HCl 150 mM NaCl 6M Gu.HCl 10% Glycerol 20 mM Imidazole pH 8, Lane7: pellet produced during the dialysis, Lane 8: Eluted with 20 mMTris.HCl 150 mM NaCl 6M Gu.HCl 10% Glycerol 250 mM Imidazole pH 8. (B)Lane 1: Sf9bacmid, Lane 2: whole cell lysate, Lane 3: pellet treatedwith 6M Gu.HCl, Lane 4: Eluted with 20 mM Tris-HCL 150 mM NaCl 6M Gu.HCl10% Glycerol 20 mM Imidazole pH 8, Lane 5: pellet produced during thedialysis, Lane 6: Eluted with 20 mM Tris-HCL 150 mM NaCl 6M Gu.HCl 10%Glycerol 250 mM Imidazole pH 8.

FIGS. 9A-B show cystine depletion of cystinotic fibroblasts by treatmentwith media from Sf9 cells expressing human cystinosin. 10 ml of mediumremoved from Sf9 cells transfected with CTNS-containing Bacmid wasdialyzed against 500 ml Ham's F12 medium for 24 hours using regeneratedcellulose dialysis tubing. After 24 hours one 500 ml medium change wasperformed, for a final effective dilution of 1/2500. The medium insidethe dialysis membrane was removed, supplemented with 15% FBS andpenicillin, streptomycin and Fungizone, and then was placed uponcystinotic fibroblasts. A control series was performed using the mediumfrom outside the dialysis tubing, which has a MW cut-off of 3500Daltons. This medium was supplemented exactly like the other medium.Three separate cystinotic cell lines, have been employed: GM00008 ishomozygous for the 57 kb deletion; GM00018 does not have a specificdefect listed on the Coriell site; GM00046 is homozygous for a 5 bpdeletion at nucleotide 545 of exon 5 of the CTNS gene (545delTCCTT).Intracellular cystine is increased. This experiment has been done atotal of five times. The cells were harvested by trypsinization at zero24, 48, and 96 hours, and tested for cystine measurement. Over thefour-day period there was a rapid and progressive decline in the cystinecontent to about 30% of control value for cells treated with thematerial inside the dialysis membrane, and little depletion from thecells treated with the media obtained from outside the dialysismembrane.

FIG. 10 shows cystine depletion of cystinotic fibroblasts by treatmentwith media from Sf9 cells not expressing human cystinosin; whenconditioned medium from non-transfected Sf9 cells was dialyzed andprepared identically to that described and placed on cultured cystinoticfibroblasts, no difference in cystine depletion was seen between thosecells treated with material from inside the dialysis membrane ascompared top that from outside the membrane.

FIG. 11 shows a graph of relative cystine depletion levels by variousportions (dialysate, dialysis fluid, sedementable fraction, supernatant)of media conditioned by CTNS-transfected or sialin-transfected cells.

FIG. 12 shows a graph of relative sialic acid depletion levels byvarious portions (dialysate, dialysis fluid, sedementable fraction,supernatant) of media conditioned by CTNS-transfected orsialin-transfected cells.

FIG. 13 shows Western blot analysis of protein expression fromcystinosin-transfected Sf9 cells. Data are consistent with expression ofhuman cystinosin in the Baculovirus infected Sf9 cells.

DEFINITIONS

As used herein the term “cystinosin replacement factor” or “CRF” refersto an agent or agents that perform the in vivo and/or in vitro functionof cystinosin. A “CRF” may replace one or more (e.g. all) of thebiological functions of endogenous wild-type cystinosin, including, butnot limited to transport of lysosomal cystine, transporting cystine fromthe lysosomal interior to the cytosol, and/or depletion of cystine fromlysosomes (e.g. cystinotic lysosomes). A “CRF” performs a biologicalfunction of cystinosin, typically by the same mechanism of action. A“CRF” comprises any compound (e.g. small molecule, polymer, etc.) and/orbiomolecule (e.g. protein, peptide, nucleic acid, macromolecule, etc.)that replaces one or more biological functions of cystinosin. A “CRF”typically replaces the function of cystinosin in a subject sufferingfrom cystinosis, with defective cystinosin function, and/or with aninadequate amount of cystinosin. A “CRF” may comprise proteins orpolypeptides with similar sequence or structure to cystinosin, variantsof cystinosin, truncated versions of cystinosin, mutated cystinosin,and/or combinations thereof. A “CRF” may comprise cystinosin (e.g.exogenously produced cystinosin).

As used herein the term “cystine depletion factor” or “CDF” refers to anagent or agents that depletes lysosomal cystine or causes a reduction inthe concentration of cystine in lysosomes (e.g. cystinotic lysosomes). ACDF may be a cystinosin replacement factor or may deplete lysosomalcystine by a different mechanism, path, or biological function. In someembodiments, a “CDF” comprises any compound (e.g. small molecule,polymer, etc.) and/or biomolecule (e.g. protein, peptide, nucleic acid,macromolecule, etc.) that directly or indirectly (e.g. by acting onanother factor (e.g. protein)) reduces the cystine concentration inlysosomes. In some embodiments, a “CDF” only reduces the cystineconcentration in cystinotic lysosomes. A “CRF” may comprise proteins orpolypeptides with similar sequence or structure to cystinosin, variantsof cystinosin, truncated versions of cystinosin, mutated cystinosin,and/or combinations thereof. A “CRF” may comprise cystinosin (e.g.exogenously produced cystinosin).

The terms “protein” and “polypeptide” refer to compounds comprisingamino acids joined via peptide bonds and may be used interchangeably.

As used herein, where “amino acid sequence” is recited herein to referto an amino acid sequence of a protein or peptide molecule. An “aminoacid sequence” can be deduced from the nucleic acid sequence encodingthe protein. However, terms such as “polypeptide” or “protein” are notmeant to limit the amino acid sequence to the deduced amino acidsequence, but include post-translational modifications of the deducedamino acid sequences, such as amino acid deletions, additions, andmodifications such as glycolsylations and addition of lipid moieties.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises coding sequences necessary for the production of an RNA,or a polypeptide or its precursor (e.g., proinsulin). A functionalpolypeptide can be encoded by a full length coding sequence or by anyportion of the coding sequence as long as the desired activity orfunctional properties (e.g., enzymatic activity, ligand binding, signaltransduction, etc.) of the polypeptide are retained. The term “portion”when used in reference to a gene refers to fragments of that gene. Thefragments may range in size from a few nucleotides to the entire genesequence minus one nucleotide. Thus, “a nucleotide sequence comprisingat least a portion of a gene” may comprise fragments of the gene or theentire gene.

The term “gene” also encompasses the coding regions of a structural geneand includes sequences located adjacent to the coding region on both the5′ and 3′ ends for a distance of about 1 kb on either end such that thegene corresponds to the length of the full-length mRNA. The sequenceswhich are located 5′ of the coding region and which are present on themRNA are referred to as 5′ non-translated sequences. The sequences whichare located 3′ or downstream of the coding region and which are presenton the mRNA are referred to as 3′ non-translated sequences. The term“gene” encompasses both cDNA and genomic forms of a gene. A genomic formor clone of a gene contains the coding region interrupted withnon-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are segments of a gene which aretranscribed into nuclear RNA (mRNA); introns may contain regulatoryelements such as enhancers. Introns are removed or “spliced out” fromthe nuclear or primary transcript; introns therefore are absent in themessenger RNA (mRNA) transcript. The mRNA functions during translationto specify the sequence or order of amino acids in a nascentpolypeptide.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequenceswhich are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers which control or influence thetranscription of the gene. The 3′ flanking region may contain sequenceswhich direct the termination of transcription, posttranscriptionalcleavage and polyadenylation.

The term “polynucleotide” refers to a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three, andusually more than ten. The exact size will depend on many factors, whichin turn depends on the ultimate function or use of the oligonucleotide.The polynucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, or a combinationthereof. The term “oligonucleotide” generally refers to a short lengthof single-stranded polynucleotide chain usually less than 30 nucleotideslong, although it may also be used interchangeably with the term“polynucleotide.”

The term “nucleic acid” refers to a polymer of nucleotides, or apolynucleotide, as described above. The term is used to designate asingle molecule, or a collection of molecules. Nucleic acids may besingle stranded or double stranded, and may include coding regions andregions of various control elements, as described below.

The term “a polynucleotide having a nucleotide sequence encoding a gene”or “a polynucleotide having a nucleotide sequence encoding a gene” or “anucleic acid sequence encoding” a specified polypeptide refers to anucleic acid sequence comprising the coding region of a gene or in otherwords the nucleic acid sequence which encodes a gene product. The codingregion may be present in either a cDNA, genomic DNA or RNA form. Whenpresent in a DNA form, the oligonucleotide, polynucleotide, or nucleicacid may be single-stranded (i.e., the sense strand) or double-stranded.Suitable control elements such as enhancers/promoters, splice junctions,polyadenylation signals, etc. may be placed in close proximity to thecoding region of the gene if needed to permit proper initiation oftranscription and/or correct processing of the primary RNA transcript.Alternatively, the coding region utilized in the expression vectors ofthe present invention may contain endogenous enhancers/promoters, splicejunctions, intervening sequences, polyadenylation signals, etc. or acombination of both endogenous and exogenous control elements.

The term “vector” refers to nucleic acid molecules that transfer DNAsegment(s) from one cell to another. The term “vehicle” is sometimesused interchangeably with “vector.”

The terms “expression vector” or “expression cassette” refer to arecombinant DNA molecule containing a desired coding sequence andappropriate nucleic acid sequences necessary for the expression of theoperably linked coding sequence in a particular host organism. Nucleicacid sequences necessary for expression in prokaryotes usually include apromoter, an operator (optional), and a ribosome binding site, oftenalong with other sequences. Eukaryotic cells are known to utilizepromoters, enhancers, and termination and polyadenylation signals.

The term “type of nucleic acid” refers to a characteristic or propertyof a nucleic acid that can distinguish it from another nucleic acid,such as a difference in sequence or in physical form, such as occurs indifferent expression vectors, or as occurs with the presence of DNA andRNA, or as occurs with the presence of linear and super-coiled DNA, oras occurs with the presence of coding regions which encode differentproteins, or as occurs with the presence of different control elements,or control elements which differ amongst themselves.

The term “transfection” refers to the introduction of foreign DNA intocells. Transfection may be accomplished by a variety of means known tothe art including calcium phosphate-DNA co-precipitation,DEAE-dextran-mediated transfection, polybrene-mediated transfection,glass beads, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, viral infection, biolistics (i.e.,particle bombardment) and the like.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell. The foreign DNApersists in the nucleus of the transfected cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes. The term“transient transfectant” refers to cells that have taken up foreign DNAbut have failed to integrate this DNA.

The term “host cell” refers to any cell capable of replicating and/ortranscribing and/or translating a heterologous gene. Thus, a “host cell”refers to any eukaryotic or prokaryotic cell (e.g., bacterial cells suchas E. coli, yeast cells, mammalian cells, avian cells, amphibian cells,plant cells, fish cells, and insect cells), whether located in vitro orin vivo. For example, host cells may be located in a transgenic animal.

The terms “transformants” or “transformed cells” include the primarytransformed cell and cultures derived from that cell without regard tothe number of transfers. All progeny may not be precisely identical inDNA content, due to deliberate or inadvertent mutations. Mutant progenythat have the same functionality as screened for in the originallytransformed cell are included in the definition of transformants.

The term “selectable marker” refers to a gene which encodes an enzymehaving an activity that confers resistance to an antibiotic or drug uponthe cell in which the selectable marker is expressed, or which confersexpression of a trait which can be detected (e.g., luminescence orfluorescence). Selectable markers may be “positive” or “negative.”Examples of positive selectable markers include the neomycinphosphotrasferase (NPTII) gene which confers resistance to G418 and tokanamycin, and the bacterial hygromycin phosphotransferase gene (hyg),which confers resistance to the antibiotic hygromycin. Negativeselectable markers encode an enzymatic activity whose expression iscytotoxic to the cell when grown in an appropriate selective medium. Forexample, the HSV-tk gene is commonly used as a negative selectablemarker. Expression of the HSV-tk gene in cells grown in the presence ofgancyclovir or acyclovir is cytotoxic; thus, growth of cells inselective medium containing gancyclovir or acyclovir selects againstcells capable of expressing a functional HSV TK enzyme.

The term “reporter gene” refers to a gene encoding a protein that may beassayed. Examples of reporter genes include, but are not limited to,luciferase (See, e.g., deWet et al., Mol. Cell. Biol. 7:725 (1987) andU.S. Pat. Nos. 6,074,859; 5,976,796; 5,674,713; and 5,618,682; all ofwhich are incorporated herein by reference), green fluorescent protein(e.g., GenBank Accession Number U43284; a number of GFP variants arecommercially available from ClonTech Laboratories, Palo Alto, Calif.),chloramphenicol acetyltransferase, β-galactosidase, alkalinephosphatase, and horse radish peroxidase.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” refers to a nucleic acid sequence that isidentified and separated from at least one contaminant nucleic acid withwhich it is ordinarily associated in its natural source. Isolatednucleic acid is present in a form or setting that is different from thatin which it is found in nature. In contrast, non-isolated nucleic acids,such as DNA and RNA, are found in the state they exist in nature. Forexample, a given DNA sequence (e.g., a gene) is found on the host cellchromosome in proximity to neighboring genes; RNA sequences, such as aspecific mRNA sequence encoding a specific protein, are found in thecell as a mixture with numerous other mRNA s which encode a multitude ofproteins. However, isolated nucleic acid encoding a particular proteinincludes, by way of example, such nucleic acid in cells ordinarilyexpressing the protein, where the nucleic acid is in a chromosomallocation different from that of natural cells, or is otherwise flankedby a different nucleic acid sequence than that found in nature. Theisolated nucleic acid or oligonucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acidor oligonucleotide is to be utilized to express a protein, theoligonucleotide will contain at a minimum the sense or coding strand(i.e., the oligonucleotide may single-stranded), but may contain boththe sense and anti-sense strands (i.e., the oligonucleotide may bedouble-stranded).

The term “purified” refers to molecules, either nucleic or amino acidsequences, that are removed from their natural environment, isolated orseparated. An “isolated nucleic acid sequence” is therefore a purifiednucleic acid sequence. “Substantially purified” molecules are at least60% free, preferably at least 75% free, and more preferably at least 90%free from other components with which they are naturally associated. Asused herein, the term “purified” or “to purify” also refers to theremoval of contaminants from a sample. The removal of contaminatingproteins results in an increase in the percent of polypeptide ofinterest in the sample. In another example, recombinant polypeptides areexpressed in plant, bacterial, yeast, or mammalian host cells and thepolypeptides are purified by the removal of host cell proteins; thepercent of recombinant polypeptides is thereby increased in the sample.

The term “sample” is used in its broadest sense. In one sense it canrefer to biological and environmental samples. Biological samples may beobtained from animals (including humans) and encompass fluids, solids,tissues, and gases. Biological samples include blood products, such asplasma, serum and the like. Environmental samples include environmentalmaterial such as surface matter, soil, water, and industrial samples.These examples are not to be construed as limiting the sample typesapplicable to the present invention.

As used herein, the term “effective amount” refers to the amount of acomposition sufficient to effect beneficial or desired results. Aneffective amount can be administered in one or more administrations,applications or dosages and is not intended to be limited to aparticular formulation or administration route.

As used herein, the term “administration” refers to the act of giving adrug, prodrug, or other agent, or therapeutic treatment (e.g.,compositions of the present invention) to a subject (e.g., a subject orin vivo, in vitro, or ex vivo cells, tissues, and organs). Exemplaryroutes of administration to the human body can be through the eyes(ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs(inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g.,intravenously, subcutaneously, intratumorally, intraperitoneally, etc.)and the like.

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent with a carrier, inert or active, makingthe composition especially suitable for diagnostic or therapeutic use invitro, in vivo or ex vivo.

The terms “pharmaceutically acceptable” or “pharmacologicallyacceptable,” as used herein, refer to compositions that do notsubstantially produce adverse reactions, e.g., toxic, allergic, orimmunological reactions, when administered to a subject.

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers including, but not limitedto, phosphate buffered saline solution, water, emulsions (e.g., such asan oil/water or water/oil emulsions), and various types of wettingagents, any and all solvents, dispersion media, coatings, sodium laurylsulfate, isotonic and absorption delaying agents, disintigrants (e.g.,potato starch or sodium starch glycolate), and the like. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants. (See e.g., Martin,Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton,Pa. (1975), incorporated herein by reference).

As used herein, the term “pharmaceutically acceptable salt” refers toany salt (e.g., obtained by reaction with an acid or a base) of acompound of the present invention that is physiologically tolerated inthe target subject (e.g., a mammalian subject, and/or in vivo or exvivo, cells, tissues, or organs). “Salts” of the compounds of thepresent invention may be derived from inorganic or organic acids andbases. Examples of acids include, but are not limited to, hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic,malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and thelike. Other acids, such as oxalic, while not in themselvespharmaceutically acceptable, may be employed in the preparation of saltsuseful as intermediates in obtaining the compounds of the invention andtheir pharmaceutically acceptable acid addition salts.

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention provides compositions and methods for providingfactor replacement therapy. In some embodiments, the present inventionprovides methods for producing one or more factors useful in factorreplacement therapy. In some embodiments, the present invention providesfactors produced through recombinant protein expression, that findutility in factor replacement therapy. In some embodiments, a factor maybe a protein (e.g. enzyme (e.g. cystinosin)), vitamin, nutrient,compound, composition, nucleic acid, small molecule, macromolecule,molecular complex, etc.

In some embodiments, the present invention provides methods for theproduction of factors for use in factor replacement therapy. In someembodiments, a subject is deficient in the endogenous production of oneor more factors (e.g. cystinosin). In some embodiments, a subjectproduces defective factor, and is thereby deficient innormal/active/functioning factor. In some embodiments, a properlyexpressed factor is rendered defective through the action of anotherfactor or factors, or by an unknown mechanism or pathway. In someembodiments, a factor deficiency (e.g. cystinosin deficiency) results ina disease, condition, and or disorder (e.g. cystinosis) in a factordeficient subject. In some embodiments, treatment of a disease ordisorder (e.g. cystinosis) is performed by replacing the defectivefactor (e.g. protein (e.g. cystinosin)) with normal/active/functioningfactor (e.g. produced exogenously (e.g. exogenously expressedcystinosin)), a variant of the factor, or another factor (e.g. relatedor unrelated). In some embodiments, replacement of, supplementing of, orcompensation for the factor for which a subject is deficient providestherapy (e.g. curative, palliative) for the related condition (e.g.symptom reduction). In some embodiments, the factor provided inreplacement therapy is the factor for which the subject is deficient(e.g. cystinosin). In some embodiments, the factor provided inreplacement therapy is not the factor for which the subject isdeficient. In some embodiments, the factor provided in replacementtherapy is related to, a product of, and/or a by-product of expressionof the factor for which the subject is deficient. In some embodiments,the factor provided in replacement therapy is a variant, mutant(e.g. >50% identity . . . >60% identity . . . >70% identity . . . >80%identity . . . >90% identity . . . >95% identity . . . >98% identity . .. >99% identity), or truncated version (e.g. >10% length of wild-type .. . >20% length of wild-type . . . >30% length of wild-type . . . >40%length of wild-type . . . >50% length of wild-type . . . >60% length ofwild-type . . . >70% length of wild-type . . . >80% length of wild-type. . . >90% length of wild-type . . . >95% length of wild-type . . . >98%length of wild-type . . . >99% length of wild-type) of the factor forwhich the subject is deficient (e.g. cystinosin). In some embodiments, afactor or factors secreted by the cells expressing the factor for whichthe subject is deficient provides a replacement or supplement for thefactor-deficient subject. In some embodiments, the factor for which thesubject is deficient is secreted by the cells expressing the factor forwhich the subject is deficient, and the secreted factor provides areplacement or supplement for the factor-deficient subject. In someembodiments, one or more factors are purified and/or isolated from theliquid media. In some embodiments, one or more factors are purifiedand/or isolated along with a plurality of other compounds andcompositions from the liquid media. In some embodiments, a mixturecomprising the desired factor or factors and additional components fromthe liquid media provide a replacement and/or supplement for a factordeficiency.

In some embodiments, the present invention provides a compositionconfigured to deplete lysosomes of cystine. In some embodiments, thepresent invention provides a composition configured to depletecystinotic lysosomes of cystine. In some embodiments, the presentinvention provides a cystine depletion factor (CDF). In someembodiments, compositions and methods of the present invention provide areduction in the level (e.g. concentration) of lysosomal cystine (e.g.in cystinotic lysosomes). In some embodiments, a CDF reduces the cystinelevel in cystinotic lysosomes. In some embodiments, a CDF reduces thecystine level in lysosomes through the same mechanism and/or pathway ascystinosin. In some embodiments, a CDF reduces the cystine level inlysosomes via an alternative mechanism and/or pathway to that utilizedby cystinosin. In some embodiments, a CDF reduces the cystine level incystinotic lysosomes by at least 50% (e.g. at least 50% . . . at least60% . . . at least 70% . . . at least 80% . . . at least 90% . . . atleast 950% . . . at least 98% . . . at least 99%). In some embodiments,CDF is cystinosin (e.g. exosome encapsulated cystinosin). In someembodiments, CDF is a factor (e.g. protein, polypeptide, nucleic acid,etc.) other than cystinosin. In some embodiments, CDF is produces and/orexcreted from cells expressing cystinosin (e.g. exogenous cystinosin).In some embodiments, a CDF is a CRF.

In some embodiments, the present invention provides a compositionconfigured to replace cystinosin in cystinosin-deficient subject,subject suffering from cystinosis, and/or subjects lacking functionalcystinosin. In some embodiments, the present invention provides acomposition configured to replace the function of a subject's endogenouscystinosin. In some embodiments, the present invention providescompositions comprising a cystinosin replacement factor (CRF). In someembodiments, a CRF is administered to a subject (e.g. subject sufferingfrom cystinosis) to provide, replace, or supplement cystinosin function.In some embodiments, CRF performs the same biological functions ascystinosin. In some embodiments, CRF acts within the same biochemicalpathways as cystinosin. In some embodiments, CRF is cystinosin (e.g.exosome encapsulated cystinosin). In some embodiments, CRF is a factor(e.g. protein, polypeptide, nucleic acid, etc.) other than cystinosin.In some embodiments, CRF is produces and/or excreted from cellsexpressing cystinosin (e.g. exogenous cystinosin). In some embodiments,a CRF is a CDF.

In some embodiments, the present invention provides exosomes (e.g.exosomes encapsulating CDF, CRF, and/or cystinosin), and methods ofpreparation, isolation, purification, administration, and use thereof.In some embodiments, exosomes comprises CDF, CRF, and/or cystinosin. Insome embodiments, exosomes comprises lysosomal trans-membrane proteins.In some embodiments, exosomes are secreted from cells. In someembodiments, exosomes are secreted from cells (e.g. Sf9 cells) that havebeen engineered (e.g. transfected, transformed, etc.) to expresscystinosin. In some embodiments, cells (e.g. Sf9 cells) that have beenengineered (e.g. transfected, transformed, etc.) to express cystinosinsecrete exosomes comprising CDF, CRF, and/or cystinosin. In someembodiments, cells that have been engineered to express cystinosingenerate exosomes comprising cystinosin. In some embodiments exosomes(e.g. exosomes encapsulating CDF, CRF, and/or cystinosin) generated fromcells are purified and/or isolated from other cellular and/or mediacomponents. In some embodiments, exosomes (e.g. exosomes encapsulatingCDF, CRF, and/or cystinosin) are purified and/or isolated from some orall other cellular and/or media components by standard methodologiesknown to those in the art, not limited to: dialysis, centrifugation,chromatography, gel electrophoresis, filtration, etc. In someembodiments, the encapsulated contents (e.g. CDF, CRF, and/orcystinosin) of an exosome is co-purified with the exosome. In someembodiments, CDF, CRF, and/or cystinosin is purified and/or isolatedfrom exosomes. In some embodiments, CDF, CRF, and/or cystinosin is notpurified from exosomes before use and/or administration. In someembodiments, exosomes comprising (e.g. encapsulating) CDF, CRF, and/orcystinosin are administered to a subject to treat and/or prevent adisease, condition or disorder (e.g. cystinosis). In some embodiments,encapsulation of CDF, CRF, and/or cystinosin within exosomes does notinhibit the effectiveness of administration of cystinosin. In someembodiments, encapsulation of CDF, CRF, and/or cystinosin withinexosomes enhances the effectiveness of administration of cystinosin. Insome embodiments, encapsulation of CDF, CRF, and/or cystinosin withinexosomes does not inhibit and/or enhances cellular uptake, subcellularlocalization, targeting to the lysosome, stability, etc. of CDF, CRF,and/or cystinosin. In some embodiments, exosomes (e.g. exosomesencapsulating CDF, CRF, and/or cystinosin) are about 50-100 nm (e.g.60-80 nm) in diameter. In some embodiments, exosomes (e.g. exosomesencapsulating CDF, CRF, and/or cystinosin) are small enough to passthrough a sterilization filter. In some embodiments, exosomes arefreeze-thaw resistant. In some embodiments, exosomes provide a deliverysystem for CDF, CRF, and/or cystinosin by preferentially fusing with theendosomal system of cystinotic fibroblasts. In some embodiments,exosomes provide a transmembrane delivery system for CDF, CRF, and/orcystinosin.

In some embodiments, the present invention provides compositions andmethods for expressing factors for use in factor replacement therapy. Insome embodiments, factors for factor replacement therapy are expressedin a usable, stable, cell permeable, and/or effective form (e.g.encapsulated in exosomes). In some embodiments, encapsulation of factorswithin exosomes provides a delivery vehicle for the factor which allowscell entry and/or subcellular localization of the factor. In someembodiments, encapsulation within exosomes provides active factors. Insome embodiments, encapsulation within exosomes provides solublefactors.

In some embodiments, the present invention provides cells which produce(e.g. express) a factor for which a subject is deficient. In someembodiments, the present invention provides cells which produce (e.g.express) a replacement factor (e.g. CRF) which performs one or more ofthe functions of the factor for which a subject is deficient. In someembodiments, cells are engineered (e.g. cloning, transformation,transfection, etc.) to produce a factor (e.g. protein) for use in thepresent invention. Techniques for engineering cells to produce a desiredfactor are understood in the art. In some embodiments, cells producingone or more factors are grown in liquid media. In some embodiments,cells secrete one or more factors into the liquid media within whichthey are grown. In some embodiments, cells secrete the factor (e.g.cystinosin), for which a subject is deficient, into the liquid media. Insome embodiments, cells secrete one or more factors other than the onefor which a subject is deficient (e.g. cystinosin) into the liquidmedia. In some embodiments, cells secrete a factor which is useful intreating a factor-deficient subject (e.g. a factor or factors other thanthe one for which the subject is deficient).

A variety of replacement factors may be generated using the methodsdescribed herein. In some embodiments, the replacement factor replacescystinosin. In some embodiments, the replacement factor replaces afactor that is insoluble or substantially insoluble. In someembodiments, the replacement factor replaces a factor including, but notlimited to, NiemannPick C disease (NPC), Sialic acid storage disease,Kufor-Rakeb syndrome, Batten Disease, etc.

In some embodiments, the present invention provides cloning a nucleicacid (e.g. gene) of interest into a vector. In some embodiments, thepresent invention provides transforming or transfecting a vector into acell and/or cell line. In some embodiments, the present inventionprovides growing cells (e.g. in liquid media) under conditions such thata protein of interest is expressed (e.g. overexpressed). In someembodiments, the present invention provides collecting the media withinwhich cells expressing a protein of interest have been grown. In someembodiments, the present invention provides isolating, concentrating,and/or purifying one or more factors, compounds, proteins, complexes,nutrients, etc. from the media within which cells expressing a proteinof interest have been grown. In some embodiments, the present inventionprovides administering an isolated/purified fraction of the media withinwhich cells expressing a protein of interest have been grown to a sample(e.g. cells) or subject deficient in the protein of interest. Isolatedmolecules or fractions may be added to other components, including, butnot limited to, pharmaceutically acceptable buffers, other therapeuticagents, etc. In some embodiments, the present invention providesadministering an isolated/purified fraction of the media within whichcells expressing a protein of interest were grown. In some embodiments,an isolated/purified fraction of the media, within which cellsexpressing a protein of interest have been grown, compensates for themissing protein of interest in a sample or subject (e.g. providestherapy for the sample or subject). In some embodiments, a portion ofthe cells, cellular components, excreted factors, and/or the media inwhich they were grown is purified and/or isolated by any acceptablemethod including but not limited to dialysis, precipitation,chromatography, gel purification, centrifugation, ultracentrifugation,sedimentation, and/or combinations thereof.

In some embodiments, the present invention provides therapy for asubject suffering from cystinosis. In some embodiments, therapy providesone or more factors (e.g. cystine depletion factors) which address theaccumulation of cystine in lysosomes caused by cystinosis. In someembodiments, the present invention provides replacement therapy for asubject suffering from cystinosis. In some embodiments, replacementtherapy provides one or more replacement factors (e.g. cystinosinreplacement factors) which perform one or more functions of the missingand/or deficient cystinosin. In some embodiments, the present inventionprovides compositions and methods to produce CDF or CRF for thetreatment of cystinosis.

In some embodiments, a cystinosin gene (e.g. wild-type, mutated,truncated, etc.) is cloned into a suitable vector, and the vector istransformed or transfected into a cell or cell line. In someembodiments, cells containing the cystinosin gene are grown in liquidmedia under conditions such that cystinosin is expressed (e.g.overexpressed). In some embodiments, cystinosin is secreted from thecells. In some embodiments, cells producing cystinosin secrete otherfactors related to the expression of cystinosin. In some embodiments,cells producing cystinosin secrete one or more factors that find use ascystinosin replacement factors and/or cystine depletion factors. In someembodiments, cells, media, and or other factors (e.g. secreted factors(e.g. exosomes, secreted cystinosin, etc.), and purified and or isolatedby suitable means. In some embodiments, the media fromcystinosin-expressing cells is collected (e.g. by centrifugation, byfiltration, etc.). In some embodiments, material secreted by cells (e.g.exosomes, cystinosin, etc.) are collected with the media. In someembodiments, the media is separated into fractions (e.g. bychromatography, by centrifugation, by ultracentrifugation, byfiltration, by affinity, etc.). In some embodiments, one or moreelements, vesicles (e.g. exosomes), compositions, compounds, proteins,etc. are isolated from the media. In some embodiments, the media ispurified away from one or more contaminants. In some embodiments, thecollected, fractionated, purified, and/or isolated media is configuredto be administered as a factor replacement therapy for cystinosis. Insome embodiments, the collected, fractionated, purified, and/or isolatedmedia is configured to be administered as a lysosomal cystine depletiontherapy for cystinosis. In some embodiments, administration of thecollected, fractionated, purified, and/or isolated media provides atherapy for cystinosis. In some embodiments, the collected,fractionated, purified, and/or isolated media comprises soluble andactive cystinosin. In some embodiments, the collected, fractionated,purified, and/or isolated media comprises cystinosin-related factors. Insome embodiments, the collected, fractionated, purified, and/or isolatedmedia comprises one or more CRF. In some embodiments, the collected,fractionated, purified, and/or isolated media comprises one or more CDF.In some embodiments, the collected, fractionated, sedimented, purified,and/or isolated media comprises factors capable of replacing, restoring,and/or compensating for endogenous cystinosin in subjects suffering fromcystinosis. In some embodiments, the present invention providesadministering isolated cystinosin as a therapy for cystinosis.

The compositions and methods of the present invention find use in thetransfection of any number of cell types. Cells may be in vitro, inculture, ex vivo, or in vivo. In some embodiments, the systems andmethods of the present invention find use in research, clinical, ordiagnostic applications. In some embodiments, the present inventionprovides compositions and methods molecular biology techniques such ascloning, protein expression, protein purification, transforming andtransfecting cells, growing cells in media, and other related methodsdescribed in the following references: Sambrook et al. (Ed.), “MolecularCloning, a Laboratory Manual (3rd edition), Cold Spring HarborPress andCold Spring Harbor, N.Y. (2001), Ausubel et al. (Ed.), “CurrentProtocols in Molecular Biology,” John Wiley & Sons Ltd., hereinincorporated by reference in their entireties.

EXPERIMENTAL Example 1 Cloning and Solubility

The human cystinosin encoding gene was subcloned into pFastBacHTA vectorand the resulted construct was transformed into E. coli strain DH10Bacto generate recombinant Bacmid. The gene was cloned into the pFastBacHTAvector at EcoR1 and Not 1 restriction sites (restriction sites areunderlined below).

(SEQ ID NO: 1) 1 GAATTCATGA TCCGTAACTG GCTGACTATC TTCATCCTGT TCCCTCTGAAGCTGGTCGAG 61 AAGTGCGAGT CCTCCGTCAG CCTCACCGTG CCTCCCGTGG TGAAGCTGGAGAACGGTAGC 121 TCCACCAACG TCAGCCTCAC CCTGCGCCCC CCACTGAACG CCACCCTGGTGATCACCTTC 181 GAGATCACTT TCCGCTCCAA GAACATCACC ATCCTGGAGC TGCCTGACGAGGTGGTCGTG 241 CCTCCTGGTG TGACTAACTC TTCTTTCCAG GTGACCTCCC AGAACGTCGGACAGCTGACC 301 GTGTACCTGC ACGGAAACCA CTCCAACCAG ACCGGACCCC GCATCCGCTTCCTCGTCATC 361 AGGTCCTCTG CTATCAGCAT CATCAACCAG GTGATCGGTT GGATCTACTTCGTGGCTTGG 421 AGCATCTCTT TCTACCCACA GGTCATCATG AACTGGAGGC GTAAGTCCGTGATCGGTCTG 481 TCCTTCGACT TCGTCGCTCT CAACCTGACC GGTTTCGTCG CTTACTCTGTGTTCAACATC 541 GGCCTCCTCT GGGTGCCCTA CATCAAGGAG CAGTTCCTCC TCAAGTACCCTAACGGTGTG 601 AACCCCGTCA ACTCCAACGA CGTGTTCTTC AGCCTGCACG CTGTCGTGCTGACCCTCATC 661 ATCATCGTCC AGTGCTGCCT GTACGAGCGT GGTGGCCAGC GCGTGTCCTGGCCTGCTATC 721 GGCTTCCTGG TCCTGGCCTG GCTGTTCGCT TTCGTCACTA TGATCGTGGCTGCTGTGGGT 781 GTGATCACCT GGCTGCAGTT CCTGTTCTGC TTCAGCTACA TCAAGCTGGCTGTCACCCTC 841 GTGAAGTACT TCCCTCAGGC TTACATGAAC TTCTACTACA AGAGCACTGAGGGTTGGTCC 901 ATCGGAAACG TGCTGCTGGA CTTCACCGGC GGCTCTTTCT CCCTGCTGCAGATGTTCCTG 961 CAGTCCTACA ACAACGACCA GTGGACCCTC ATCTTCGGAG ACCCCACTAAGTTCGGACTG 1021 GGTGTGTTCT CTATCGTCTT CGACGTGGTG TTCTTCATCC AGCACTTCTGCCTGTACCGC 1081 AAGCGCCCCG GATACGACCA GCTCAACTAA TAAGCGGCCG CThe positive colonies were selected by Gm, Tet and Kan, followed by PCRidentification (SEE FIG. 1). The recombinant Bacmid was extracted andthen transfected into Sf9 cells to generate the baculovirus, usingCELLFECTIN (INVITROGEN), and incubated in HyQ liquid medium for 5 daysat 27° C. after the cells were swollen. The supernatant was collected bycentrifugation and designated as P0 virus. The cell pellet was used todetect the protein expression by SDS-PAGE and western blot analysisusing monoclonal anti-His antibody. Target protein not visualizedthrough western blot (SEE FIG. 2).

Multiplicity of infection (MOI) optimization was performed in sf9 cells.10 ml 2×10⁶/ml sf9 cells were infected at the MOI of 1, 2, 5, 10 and 20,and each 1 ml cell pellet was collected at 1 dpi, 2 dpi, 3 dpi and 4dpi. The cells were analyzed by SDS-PAGE and western blot (SEE FIG. 3),and the result demonstrates that the protein level is highest 3 dpi.

Solubility test of extracted cells demonstrates the protein is insolublein all conditions tested (SEE FIGS. 4 and 5). The protein was insolubleeven in the presence of 8M urea (SEE FIG. 6). The protein was alsoinsoluble in SKL and thiourea (SEE FIG. 7). 6M Gu.HCl was used for theextraction and purification, no obvious target protein was obtained fromthe SDS-PAGE analysis, and the target protein precipitated during thedialysis in 20 mM imidazole elution from the western blot result (SEEFIG. 8).

Example 2 Replacement Therapy

Production of Cystinosin protein in a Baculoviral expression system isaccomplished using GenScript, or another commercial source.

Purification of the soluble factor causing cystine depletion: thebioactive material which produces cystine depletion (SEE FIGS. 9 and 10and Table 1) is separated by electrophoresis on 10-20% Tris-Tricinedenaturing-SDS gels (Biorad; Cat #161-1162), blotted onto PVDF membranes(Biorad; Cat #162-0239), and the His epitope in the fusion-cystinosinprotein is detected using an anti-His antibody, as performed byGeneScript (see attachment), as well as the anti-cystinosin mousemonoclonal antibody M09, clone 5G6 (Abnova/Novus Biologicals, Littleton,Colo.; Cat # H00001497-M09); primary antibody hybridization signals aredetected using a rabbit anti-mouse HRP-conjugated secondary antibody(Chemicon International, Temecula, Calif.; Cat # AP 124P) andchemiluminiscent detection (ChemiLucent™ western blot detection system;Chemicon International; Cat #2600). 6×His tagged-cystinosin is purifiedby Ni-NTA affinity column purification (Qiagen Inc; Cat #31014).

TABLE 1 Cystine Depletion by CTNS-transfected Sf9 Media Time (hours)GM00008 GM00008 GM00046 GM00018 GM00008 Average StDev t-test nmolscystine per mg protein by trial  0 4.56 3.48 2.99 3.26 3.80 3.62 0.54 NA24 3.68 1.71 2.36 2.67 4.14 2.91 0.88 0.660 48 1.42 1.21 2.65 1.88 2.221.88 0.52 0.004 96 0.53 1.01 2.10 0.93 1.00 1.11 0.52 0.021 24 (control)NA 2.77 2.59 3.23 4.08 3.17 0.58 48 (control) NA 3.09 3.43 3.15 3.533.30 0.18 96 (control) NA 2.29 2.70 2.48 4.36 2.96 0.82 as fraction oft-0  0 1.00 1.00 1.00 1.00 1.00 1.00 0.00 NA 24 0.81 0.49 0.79 0.82 1.090.80 0.21 0.286 48 0.31 0.35 0.89 0.58 0.58 0.54 0.23 0.009 96 0.12 0.290.70 0.29 0.26 0.33 0.22 0.008 24 (control) NA 0.80 0.87 0.99 1.07 0.930.12 48 (control) NA 0.89 1.15 0.97 0.93 0.98 0.11 96 (control) NA 0.660.90 0.76 1.15 0.87 0.21Purified protein is quantified using the BCA Protein Assay Kit (PierceBiotechnology, Cat #23225. If necessary, the N-terminal 6×His tag and V5epitopes is removed from the fusion protein by TEV protease digestionbefore use in further experiments. Purified cystinosin protein isanalyzed and its sequence verified by peptide sequencing in the peptidecore at the University of Michigan.

A C-terminal fusion-cystinosin expression construct, by cloning the CTNSORF into the BaculoDirect™ Baculovirus C-term expression vector(Invitrogen; Cat #12562-013), or expression cystinosin in anothersystem, such as the yeast Pichia pastoris, or mammalian cells is alsocontemplated.

Quantitation of purified His-tagged cystinosin is performed bycomparative densitometry. Aliquots of purified cystinosin is run on4-20% Tris-Tricine denaturing-SDS gels (Biorad; Cat #161-1162), alongwith the Smart His-tagged Protein Standard (GenScript Cat # MM0904-100),which contains several highly purified His-tagged proteins at knownconcentrations. After electrophoresis, the protein is blotted onto PVDFmembranes (Biorad; Cat #162-0239), and the hexahistidine tags in theseparated proteins are visualized using the His-Detector™ Western BlotKit, HRP Chemiluminescent kit (KPL Inc, Cat #24-00-02). This kitprovides sensitive, reproducible labeling of 6-His-tagged proteins thatis less subject to variations in signal intensity than anti-His tagantibodies, and hence can provide better quantitation. Cystinosinconcentration is estimated by comparative densitometry of cystinosinversus standard protein band intensities, using a BioRad ChemiDoc XRSsystem, available in the Department of Pediatrics Research core.

The putative soluble isoform which is non-dialyzable, and which causesthe observed cellular cystine depletion, may be the product of analternative splice site, recognized by the Sf9 cells. This isoform isidentified by use of both anti-his and anti-cystinosin antibodies, aswell as by Northern blot of message derived from the transfected Sf9cells. Northern blotting is carried out using cell pellets from thetransfected Sf9 cells using the NorthernMax kit from Ambion (catalognumber: A1940). RNA probes are made via RT-PCR using commerciallyavailable primers to CTNS (Qiagen, catalog numbers: QT00999257,QT00999264; QIAGEN OneStep RT-PCR Kit, catalog number: 210210).

Measurement of the effect of Baculovirus-produced cystinosin on thecystine content of cystinotic fibroblasts is performed using multipleseparate lines of cultured diploid epithelial fibroblasts (cystinoticand normal). The exocytosis rate in such cells was determined byfollowing the loss of a membrane-impermeant marker using ³H-mannitol. Itwas estimated that over a 24 hour period in tissue culture fibroblastswill pinocytose approximately 13 μl/10⁶ cells, and lose via exocytosis,approximately 60% of the initial lysosomal contents. Using data from asuccessful clinical trial treating patients with another lysosomalstorage diseases, MPS II, at a starting dose of idursulfase of 0.15mg/kg every other week, and assuming that the infused protein isinitially equilibrated in the vascular space for uptake by the liver andother tissues, it was calculated that the concentration initiallyachieved in the circulation would be would be 1.88 mg/1 of idursulfase(assuming a vascular volume of 8%). This results in initial delivery of1.88 ng/μl×13 μl/106 cells/24 hrs=˜24 ng of cystinosin/million cells/24hrs. If cystinosin behaves like a membrane impermeant marker, about 60%would be lost in this interval, hence a minimum estimate of cystinosinretention will be 24×0.4=˜10 ng/10⁶ cells/24 hrs.

Example 3 CDF Characterization

Experiments were conducted during development of embodiments of thepresent invention to determine the capacity of CTNS-transfectedSpodoptera conditioned medium, sialin transfected media, or putativeexosomes prepared from each medium, to deplete either cystine incystinotic cells, or sialic acid in ISSD cells after 96 h exposure.

Media was conditioned with either CTNS-transfected Spodoptera or sialin.The cells and media were either (1) dialyzed against a 3500 molecularweight cutoff membrane, and the dialysis fluid and dialysate were bothcollected; or (2) the media was separated into sedimentable andsupernatant fractions by ultracentrifugation at 140,000×g of each mediumfollowed by resuspension of the sedimentable fraction in Ham's F12tissue culture medium. ISSD cells were exposed to the dialysis fluid,dialaysate, sedimentable fraction, or supernatent for 96 hours, and thecystein concentration was measured to determine the capacity of each todeplete cystine from the cells. Cystine content was normalized to themean of the two control plates (3.2 nmol/mg protein) and set at 100%.

At 96 h, the CTNS-transfected dialysate caused a decline in the cellcystine content to about 33% of control, a value essentially equaled bythe sedimentable fraction of that medium (SEE FIG. 11). None of theother conditions caused greater than 50% depletion at 96 h. These datademonstrate that the CDF resides in the dialysate of CTNS-transfectedSpodoptera cells and can be sedimented by 140,000×g. The supernatant,above the sedimentable fraction, does not cause cystine depletion,consistent with CDF being contained in exosomes. No significant cystinedepletion occurred in cells incubated with sialin-transfected medium orits sedimentable fraction. However, the dialysate and sedimentablefraction of the sialin-transfected medium we both able to deplete sialicacid in cells, while the other conditions were unable to do so (SEE FIG.12).

Example 4 Western Blot Analysis

Experiments were conducted during development of embodiments of thepresent invention to confirm the expression of human cystinosin in cellstransfected with the gene for cystinosin (CTNS). A Western blot was madeof cell lysate from Sf9 cells infected with the CTNS-containingbaculovirus made by Genscript (SEE FIG. 13).

Sf9 cells were grown in 60 mm plates and infected at multiplicity ofinfection (MOI) of zero, 0.1, and 1.0 and then harvested 4 days postinfection by scraping and resuspended in 4×SDS Laemmli buffer. Afterbrief sonication, the samples were boiled at 100° C. for 5 minutes.SDS-PAGE was performed using 10 μL from these samples in each lane. Thegel was run was at 120V constant voltage at 64 mA-21 mA, for 107minutes. Gel transfer was done at 100V constant, 0.64 A-0.85 A mA forone hour. SC100703 mouse mAb against human cystinosin was used at aconcentration of 1:100 with secondary Promega HRP goat anti-mouse IgG,at a concentration of 1:2000. Blocking and Ab incubation buffer were 5%NFDM in PBST and ECL-plus from GE Healthcare. The blot was imaged on aTyphoon Trio scanner. A band, slightly larger than 37 kDa (arrow), isvisible only in the Baculovirus infected samples (i.e.cystinosin-transfected samples). The MW of this band is the approximateMW of cystinosin with 367 aminoacids. This band is absent in thenon-CTNS transfected Sf9 cells (control), and shows an increase indensity with MOI (1.0 MOI>0.1 MOI), indicating expression of thetransgene. The data demonstrate expression of a polypeptide consistentwith expression of human cystinosin in the Baculovirus infected Sf9cells.

All publications and patents mentioned in the present application and/orlisted below are herein incorporated by reference. Various modificationand variation of the described methods and compositions of the inventionwill be apparent to those skilled in the art without departing from thescope and spirit of the invention. Although the invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the relevant fields are intended to be within the scope ofthe following claims.

REFERENCES

The following references are herein incorporated by reference in theirentirety:

-   Gahl W A, Thoene J G, Schneider J A: Cystinosis. New Eng J Med.    2002; 347:111-121.-   Gene Therapy Patient Dies, Trial Shutdown. Associated Press,    7/26/07.-   R. J. Anderson, D. Cairns, W. A. Cardwell, M. Case, P. W.    Groundwater, A. G. Hall, L. Hogarth, A. L. Jones, O. Meth-Cohn, P.    Suryadevara, A. Tindall & J. G. Thoene: Design, Synthesis and    Initial In Vitro Evaluation of Novel Prodrugs for the Treatment of    Cystinosis. Letters in Drug Design & Discovery 3, 336-345, 2006-   Thoene J A Review of the Role of Enhanced Apoptosis in the    Pathophysiology of Cystinosis Mol Genet Metab. 2007: 92, 292-298.-   Palacin M, Goodyear G, Nunes V, Gasparini P. Cystinuria in C.    Scriver, A. Beaudet, W. Sly, D. Valle, eds. The Metabolic and    Molecular Bases of Inherited Disease, 8^(th) edition, McGraw Hill,    4909-4932, 2001.-   Town M, Cherqui S, Attard M, Forestier L, Whitmore S A, Callen D F,    Gribouval O, Broyer M, Bates G P, van't Hoff W, Antignac C. A novel    gene encoding an integral membrane protein is mutated in    nephropathic cystinosis. Nature Genet 18: 319-324, 1998.-   Thoene, J., Oshima, R., Crawhall, J., Olson, D., & Schneider, J:    Cystinosis: Intracellular Cystine Depletion by Aminothiols in Vitro    and in Vivo. J. Clin. Invest. 1976; 58:180-189.-   Shotelersuk, V., Larson, D., Anikster, Y, McDowell, G., Lemons, R.,    Bernardini, I., Guo, J., Thoene, J., Gahl, W. CTNS mutations in an    American-based population of cystinosis patients. Am Journal of    Human Genetics, 1998; 63:1352-1362.-   Muenzer J, Gucsavas-Calikoglu M, McCandless S, Schuetz T, Kimura A.    A phase I/II clinical trial of enzyme replacement therapy in    mucopolysacchardidosis II (Hunter syndrome) Mol Gen and Metab 2007,    90: 329-337.-   Lemons, R., Forster, S. and Thoene, J. Protein microinjection by    protease permeabilization of fibroblasts. Anal. Biochem. 1988; 172:    219-227.-   Pisoni, R. L., Acker, T. L., Lemons, R. M., Lisowski, K. M., and    Thoene, J. G. A cystine-specific lysosomal transport system provides    a major route for the delivery of thiol to human fibroblast    lysosomes: Role in supporting lysosomal proteolysis, J. Cell Biol.,    1990; 110:327-335.-   Lowry O, Roseborough N, Farr A, Randall R Protein measurement with    the Folin phenol reagent 1951 J Biol Chem 193; 265-275.-   Shotelersuk, V., Larson, D., Anikster, Y, McDowell, G., Lemons, R.,    Bernardini, I., Guo, J., Thoene, J., Gahl, W. CTNS mutations in an    American-based population of cystinosis patients. Am Journal of    Human Genetics, 1998; 63:1352-1362.

We claim:
 1. A composition comprising extracellular vesicles, saidextracellular vesicles comprising a recombinant lysosomal transmembraneprotein, wherein the extracellular vesicles are produced by transfectionof Spodoptera cells with baculovirus encoding the lysosomaltransmembrane protein.
 2. The composition of claim 1, wherein saidrecombinant lysosomal transmembrane protein comprises a recombinanthuman lysosomal transmembrane protein.
 3. The composition of claim 2,wherein the recombinant human lysosomal transmembrane protein isrecombinant human sialin.
 4. The composition of claim 1, wherein saidcomposition is obtained from a liquid media in contact with saidtransfected cells.
 5. The composition of claim 1, wherein saidrecombinant lysosomal transmembrane protein is within the extracellularvesicles.
 6. The composition of claim 1, further comprising apharmaceutically acceptable buffer.
 7. The composition of claim 3,wherein said recombinant human sialin functions as a replacement for anendogenous human sialin protein in a subject suffering from sialic acidstorage disease.
 8. A method for treating sialic acid storage disease,comprising: administering a composition of claim 3 to a subjectsuffering therefrom.
 9. The composition of claim 1, wherein theSpodoptera cells are Sf9 cells.